This invention relates to charged lipid compositions. In one aspect, this invention relates to negatively charged (anionic) lipid compositions, and the use of these lipid compositions for changing lipoprotein charge in vivo to clear cholesterol and other substances from the blood stream. In another aspect, this invention relates to positively charged (cationic) lipid compositions, and the use of these compositions to prolong drug activity.
Atherosclerosis leading to coronary vascular disease is a primary cause of mortality in the developed world. Atherosclerotic risk has been shown to be directly related to elevated plasma cholesterol levels. In plasma about 70% of cholesterol is esterified to long-chain fatty acids to form cholesteryl esters and these cholesteryl esters are bound to plasma lipoproteins. The lipoproteins involved in the transport of cholesterol and cholesteryl esters include low density lipoprotein (LDL), high density lipoprotein (HDL), and very-low density lipoprotein (VLDL).
While high levels of cholesterol associated with LDL have been linked to atherosclerotic risk (Schaefer et al., 1995), high HDL cholesterol levels may be protective against the development of heart disease (Miller et al., 1977). As a result there has been significant effort to develop therapies which effectively reduce the level of LDL cholesterol and raise the level of HDL cholesterol within an animal. HDL may play an anti-atherogenic role by promoting the clearance of cholesterol from the body (Eisenberg, 1984). Furthermore, Schwartz et al. (1978) disclose that cholesterol in HDL is specifically targeted for excretion from the body by the liver in the form of bile. However, current therapies directed to reduce the level of LDL cholesterol and raise the level of HDL cholesterol have not met with success.
The factors that regulate cholesterol flux to the liver are poorly understood but may involve two distinct systems; a cellular sterol regulatory system and an intravascular transport system. Excess extrahepatic cholesterol may be transported in HDL particles to the liver for excretion (Glomset, 1968). HDL has also been shown to be able to adsorb cholesterol and cholesteryl esters (CE) from cell membranes (Phillips et al., 1998). In addition, a second sterol transport pathway may include transfer of cholesterol from HDL to the rapidly turning over VLDL lipoprotein pool, followed by clearance of cholesterol by the liver (Tall, 1998).
The mechanism of intravascular sterol transport is also poorly understood, but may involve the concerted action of multiple proteins and enzymes. Two enzymes thought be involved in intravascular sterol transport is are lecithin:cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP). At present it is thought that LCAT may form a concentration gradient to move sterol into and through the blood plasma compartment by promoting the conversion of free cholesterol (FC) to cholesterol esters (CE) on HDL particles (Jonas, 1987). CETP may then promote this lipid flux by moving the newly formed CE from HDL to an apoB containing lipoprotein pool (Lagrost, 1997).
All lipoprotein classes exhibit a net negative charge, due to both the apolipoprotein composition and its content of bound charged lipids (Davidson et al., 1994). However, individual bound lipids forming part of a lipoprotein can contribute either a net positive or a negative charge, or no charge at all, to the lipoprotein. Some phospholipids, when unbound, are negatively charged, some are positively charged, and some are electrically neutral. Examples of negatively charged phospholipids are phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and phosphatidic acid. An example of an electrically neutral phospholipid is phosphatidylcholine. An example of a positively charged (cationic) phospholipid is dioleoyl trimethylammonium propane.
Williams U.S. Pat. No. 6,079,416 teaches administration of large liposomes containing phospholipids substantially free of sterols to treat hypercholesterolemia. Parker et al U.S. Pat. No. 5,614,507 teaches the injection of a bolus of phospholipid, with or without another electrically neutral lipid, to treat endotoxemia. However, the phospholipid compositions disclosed by Williams and Parker et al. are not electrically charged, so they would not act to change the electric charge of lipoproteins within the bloodstream. Instead, they act as a simple adsorbant to pick up and clear cholesterol or endotoxin from the blood.
U.S. Pat. No. 5,652,339 (Learch et al) and U.S. Pat. No. 5,128,318 (Levine et al) disclose the preparation of reconstituted high density lipoprotein (rHDL) particles, and suggest that rHDL particles may be used for drug administration and for treating diseases connected to lipids and lipodal substances. These rHDL particles said to be useable both in vivo or in vitro for removing lipid soluble materials (e.g. cholesterol, endotoxins) from cells or body fluids and aid in the treatment of hyperlipidemia and coronary atherosclerosis. Although, all lipoprotein classes exhibit a net negative charge as discussed above, these two patents teach nothing to increase the charge from that present in normal HDL.
Phosphatidylinositol (PI) is a negatively charged phospholipid found in all classes of lipoproteins and accounts for approximately 4% of the total phospholipid (PL) in HDL (Davidson et al., 1994). Incubation of PI with plasma or with isolated HDL, LDL or VLDL in vitro has shown that all of these lipoproteins can spontaneously absorb PI. However, little is known of what affects, or regulates, the amount of PI in different lipoprotein classes.
There is a need for novel compositions capable of enhancing hepatic clearance of lipoprotein particles thereby lowering cholesterol and tissue cholesterol, endotoxins and other lipid-soluble compounds such as some viruses and bacteria that associate with lipoprotein particles in vivo. There is also a need for methods to make use of such compositions. In particular, there is a need for such compositions, and methods for their use, which will preferentially lower the cholesterol associated with LDL. There is also a need for compositions, and methods for their use, which will retain drugs which associate with lipoproteins in the bloodstream, to increase the duration of the efficacy of such drugs.
The present invention provides negatively charged (anionic) lipid compositions that, when administered to an animal or subject, result in an increase in the in vivo lipoprotein negative electric charge. Associated with the increase in in vivo lipoprotein negative charge is an enhanced hepatic clearance of lipoprotein particles. The clearance of lipoprotein particles can be used for the clearance of cholesterol and has significant anti-atherogenic consequences. It can also be used to remove bacteria, endotoxin and viruses which associate with lipoproteins from the bloodstream, and for the treatment of lipid-associated diseases.
The invention also provides positively charged (cationic) lipid compositions which, when administered to an animal or subject, result in a decrease of the in vivo lipoprotein negative electric charge. Associated with the decrease in in vivo lipoprotein negative charge is a slowing of hepatic clearance of lipoprotein particles. This effect can be used to delay clearance of drugs which are associated with lipoproteins, thereby prolonging the efficacy of such drugs.
The present invention also comprises a pharmaceutical composition comprising a synthetic or naturally occurring negatively charged (anionic) phospholipid formulated into a dosage form for administration to a subject. If desired, the composition may comprise an admixture of two or more negatively charged phospholipids. The pharmaceutical composition is capable of mediating the level of lipid-associated compounds within an animal or subject. The invention also comprises the use of a negatively charged phospholipid composition as defined above for the production of a medicament useable to enhance clearance of cholesterol from the blood stream and cause the reduction in blood LDL and VLDL cholesterol levels.
The pharmaceutical compositions as discussed above can be provided in dosage forms comprising unilamellar vesicles multilamellar vesicles, multilamellar sheets, dispersions, micellar solutions, emulsions, microemulsions, pure lipid mixtures or any combination of these structures.
The present invention also relates to compositions as discussed above, wherein the dosage form is administered orally or as an injection intranasally or transdermally. In addition the composition may further comprise at least one pharmaceutically acceptable carrier.
The present invention also comprises an orally-administered food additive which comprises a charged phospholipid. When the charged phospholipid is a negatively charged phospholipid, the food additive is capable, when injested by a subject, of reducing the amount of cholesterol in the subject""s bloodstream. A particularly preferred food additive comprises phosphatidylinosotol.
The present invention also relates to preferred compositions of those discussed above, wherein the charged phospholipid is a negatively charged phospholipid selected from phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and/or phosphatidic acid or mixtures thereof, and particularly preferred compositions wherein the negatively charged phospholipid is phosphatidylinositol.
The invention also relates to a process for enhancing clearance of lipoprotein particles in vivo by administration of at least one negatively charged phospholipid. By increasing lipoprotein clearance, lipid-soluble compounds that associate with lipoprotein particles are also effectively removed. For example, as described herein, administration of charged phospholipid results in sterol mobilization into bile and excretion in faeces. Removal can therefore be accomplished of cholesterol, endotoxins, or lipoprotein-associated bacteria or virus particles. Without wishing to be bound by theory, it is thought that the administration of a pharmaceutical charged lipid composition to an animal or subject lowers serum levels of total cholesterol in the animal or subject, and inhibits the conversion of cholesterol to cholesteryl esters. Lipoprotein charge thus plays a role in regulating serum levels of lipoprotein-associated compounds, for example but not limited to free cholesterol and the like.
Also according to the invention, there is provided a method of lowering the level of cholesterol associated with LDL within a subject, the method comprising administering to the subject an effective amount of a composition comprising an anionic phospholipid composition.
Another aspect of the present invention pertains to the enhanced mobilization of cellular sterol, and the promotion of rapid clearance of both FC and CE from the plasma compartment, following the administration of a negatively charged phospholipid, (in a particularly preferred embodiment, PI) to a subject. Without wishing to be bound by theory, it is proposed that lipoprotein charge can affect cholesterol transport and that this process can be selectively manipulated by manipulating lipoprotein charge.
Other aspects of the invention provide methods of removing endotoxins, bacteria and virus particles, and treating lipid-associated diseases within an animal, including hyperlipidemia and atherosclerosis, by administering a negatively charged phospholipid to the animal or subject.
Another aspect of the invention provides a method for slowing the removal of lipoproteins, and drugs which associate with lipoproteins, from the bloodstream of an animal or subject by administering a positively charged phospholipid to such animal or subject. The invention also comprises compositions of positively charged phospholipids useful for such methods.